Protein arginine methylation is a rapidly growing field with increasingly recognized importance in cancer, but almost nothing is known about this mechanism in bone. This proposal is based on novel discoveries on the arginine methylation of Runx2. We have identified two protein arginine methyltransferases that methylate Runx2 on four defined arginine residues. By mutating these residues to alanine that mimic the methylation- deficient state, we have demonstrated the requirement for Runx2 arginine methylation in Runx2-mediated cancer cell motility. Here, we will investigate Runx2 arginine methylation in the context of its canonical role in osteoblast differentiation and bone formation. Aim 1 is to characterize the temporal arginine methylation of Runx2 in osteogenic cell culture models. We will also compare the osteogenic potentials of WT Runx2 versus unmethylatable mutant Runx2, and the consequences of manipulating methyltransferases that mediate Runx2 methylation. Aim 2 addresses the role of Runx2 arginine methylation in modulating the physical and functional interaction with BMP-Smads pathway. Finally, Aim 3 is to define the arginine methylation of Runx2, and the expression of methyltransferases that mediate Runx2 methylation during skull development in vivo, and to compare the osteogenic potential of WT versus unmethylatable Runx2 in calvarial organ cultures. Overall, this proposal addresses the hypothesis that arginine methylation is required for Runx2 activity during osteogenic differentiation and essential for BMP-Smad signaling response. Additionally, we have incorporated in the study design genomic and proteomic analyses to facilitate unbiased discoveries of Runx2 protein-protein interactions, Runx2 genomic targets and Runx2-regulated genes that are impacted by its arginine methylation. In the long run, we envision translation of knowledge acquired in this exploratory project to improve the understanding of bone development, homeostasis and regeneration, and provide novel therapeutic approaches to tackle skeletal disorders.